阅读说明：本技术 区分细菌感染和病毒感染的多重横向流测定物 (Multiple lateral flow assay to distinguish between bacterial and viral infections ) 是由 任汇淼 杨健 G·刘 于 2019-01-24 设计创作，主要内容包括：本文描述的横向流测定装置、系统和方法测量样品中多种目标分析物的浓度,并且可以确定多种目标分析物的精确浓度,其中一种或多种目标分析物在样品中以高浓度存在和其中一种或多种目标分析物以低浓度存在。当在单次施加中将单个样品施加至单个横向流测定物时,包括当第一目标分析物在单个样品中以单个样品中第二目标分析物的浓度的百万分之一存在时,可以确定多种分析物中每种的精确浓度。(The lateral flow assay devices, systems, and methods described herein measure the concentration of multiple target analytes in a sample, and can determine the precise concentration of multiple target analytes, where one or more target analytes are present in a high concentration in the sample and where one or more target analytes are present in a low concentration. When a single sample is applied to a single lateral flow assay in a single application, including when a first target analyte is present in a single sample as part per million of the concentration of a second target analyte in a single sample, the precise concentration of each of the plurality of analytes can be determined.)

1. A method of detecting a first target analyte and a second target analyte present at different concentrations in a sample, the method comprising: providing a lateral flow assay comprising:

a first complex coupled to a flow path of the lateral flow assay, the first complex comprising a label, an antibody or fragment thereof that specifically binds the first analyte, and the first analyte,

a labeled second antibody or fragment thereof attached to the flow path and configured to specifically bind to the second analyte,

a first capture zone downstream of the first complex, the first capture zone comprising a first immobilized capture agent specific for the first analyte, and

a second capture zone located downstream of the labeled second antibody or fragment thereof and comprising a second immobilized capture agent specific for the second analyte;

applying the sample to the first complex and the labeled second antibody or fragment thereof;

binding the second analyte to the labeled second antibody or fragment thereof to form a second complex;

flowing the fluid sample and the first complex to the first capture zone, wherein the first analyte in the fluid sample and the first complex compete for binding to the first immobilized capture agent in the first capture zone;

flowing the second complex in the flow path to the second capture zone and binding the second complex to the second immobilized capture agent in the second capture zone; and

detecting a first signal from the first complex bound to the first immobilized capture agent in the first capture zone and a second signal from the second complex bound to the second immobilized capture agent in the second capture zone.

2. The method of claim 1, wherein the first target analyte is present in the sample at a concentration that is about six orders of magnitude greater than the concentration of the second target analyte present in the sample.

3. The method of claim 1, wherein the first analyte of interest is present in the sample at a concentration between 1 and 999 μ l/ml and the second analyte of interest is present in the sample at a concentration between 1 and 999 pg/ml.

4. The method of claim 1, wherein the first target analyte is present in the sample at a concentration at least one order of magnitude greater than a concentration of the second target analyte present in the sample, the order of magnitude comprising one order of magnitude, two orders of magnitude, three orders of magnitude, four orders of magnitude, five orders of magnitude, six orders of magnitude, seven orders of magnitude, eight orders of magnitude, nine orders of magnitude, or ten orders of magnitude.

5. The method of claim 1, further comprising correlating the first signal with a concentration of the first target analyte present in the sample, and correlating the second signal with a concentration of a second target analyte present in the sample.

6. The method of claim 1, wherein the first signal detected by the first complex bound to the first immobilized capture agent in the first capture zone decreases as the concentration of the first analyte in the sample decreases, and wherein the second signal detected by the second complex bound to the second immobilized capture agent in the second capture zone increases as the concentration of the second target analyte in the sample increases.

7. The method of claim 1, further comprising detecting a third target analyte in the sample, wherein the lateral flow assay comprises:

a labeled third antibody or fragment thereof attached to the flow path and configured to specifically bind to the third analyte; and

a third capture zone located downstream of the labeled third antibody or fragment thereof and comprising a third immobilized capture agent specific for the third analyte.

8. The method of claim 7, further comprising:

applying the sample to the labeled third antibody or fragment thereof;

binding the third analyte to the labeled third antibody or fragment thereof to form a third complex;

flowing the third complex in the flow path to the third capture zone and binding the third complex to the third immobilized capture agent in the third capture zone; and

detecting a third signal from the third complex bound to the third immobilized capture agent in the third capture zone.

9. The method of claim 8, further comprising correlating the first signal, the second signal, and the third signal to a concentration of the first analyte, a concentration of the second analyte, and a concentration of the third analyte, respectively, in the sample.

10. The method of claim 9, further comprising indicating disease symptoms, non-disease symptoms, or no symptoms based on the respective concentrations of the first analyte, the second analyte, and the third analyte.

11. The method of claim 10, wherein the disease condition is a viral infection or a bacterial infection, and wherein the non-disease condition is inflammation.

12. The method of claim 1, wherein the first target analyte comprises a C-reactive protein (CRP) and the second target analyte comprises a TNF-related apoptosis-inducing ligand (TRAIL).

13. The method of claim 7, wherein the third target analyte comprises interferon gamma-induced protein 10 (IP-10).

14. The method of claim 1, wherein the sample is a whole blood sample, a venous blood sample, a capillary blood sample, a serum sample, or a plasma sample.

15. The method of claim 1, wherein the sample is not diluted prior to applying the sample to the lateral flow assay.

16. A lateral flow assay configured to detect a first target analyte and a second target analyte present at different concentrations in a fluid sample, the lateral flow assay comprising:

a first complex coupled to a flow path of the lateral flow assay, the first complex comprising a label, an antibody or fragment thereof that specifically binds the first analyte, and the first analyte;

a labeled second antibody or fragment thereof attached to the flow path and configured to specifically bind to the second analyte;

a first capture zone downstream of the first complex, the first capture zone comprising a first immobilized capture agent specific for the first analyte; and

a second capture zone located downstream of the labeled second antibody or fragment thereof and comprising a second immobilized capture agent specific for the second analyte.

17. The lateral-flow assay of claim 16, wherein the first complex and the labeled second antibody or fragment thereof are detached from the flow path of the lateral-flow assay when the fluid sample is applied to the first complex and the labeled second antibody or fragment thereof.

18. The lateral flow assay of claim 17, wherein when the fluid sample is applied to the first complex and the labeled second antibody or fragment thereof, the labeled second antibody or fragment thereof binds to the second analyte to form a second complex.

19. The lateral flow assay of claim 18, wherein, upon application of the fluid sample, the first immobilized capture agent in the first capture zone competitively binds to the first complex and the first analyte in the fluid sample that have flowed to the first capture zone.

20. The lateral flow assay of claim 19, wherein, upon application of the fluid sample, the second immobilized capture agent in the second capture zone binds to the second complex in the fluid sample that has flowed to the second capture zone.

21. The lateral flow assay of claim 20, wherein a first signal is emitted from the first complex bound to the first immobilized capture agent in the first capture zone and a second signal is emitted from the second complex bound to the second immobilized capture agent in the second capture zone.

22. The lateral flow assay of claim 21, wherein the first signal emitted by the first complex bound to the first immobilized capture agent in the first capture zone decreases as the concentration of the first analyte in the fluid sample decreases, and wherein the second signal emitted by the second complex bound to the second immobilized capture agent in the second capture zone increases as the concentration of the second target analyte in the fluid sample increases.

23. The lateral flow assay of claim 16, wherein the first target analyte is present in the sample at a concentration that is about six orders of magnitude greater than the concentration of the second target analyte present in the sample.

24. The lateral flow assay of claim 16, wherein the first analyte of interest is present in the sample at a concentration between 1 and 999 μ l/ml and the second analyte of interest is present in the sample at a concentration between 1 and 999 pg/ml.

25. The lateral flow assay of claim 16, wherein the first target analyte is present in the sample at a concentration at least one order of magnitude greater than the concentration of the second target analyte present in the sample, the order of magnitude comprising one order of magnitude, two orders of magnitude, three orders of magnitude, four orders of magnitude, five orders of magnitude, six orders of magnitude, seven orders of magnitude, eight orders of magnitude, nine orders of magnitude, or ten orders of magnitude.

26. The lateral flow assay of claim 16, wherein the first target analyte comprises a C-reactive protein (CRP) and the second target analyte comprises a TNF-related apoptosis-inducing ligand (TRAIL).

27. The lateral flow assay of claim 16, wherein the fluid sample is a whole blood sample, a venous blood sample, a capillary blood sample, a serum sample, or a plasma sample.

28. The lateral flow assay of claim 16, wherein the sample is not diluted prior to application to the lateral flow assay.

29. An assay test strip, comprising:

a flow path configured to receive a fluid sample;

a sample receiving zone connected to the flow path;

a detection zone connected to the flow path downstream of the sample receiving zone, the detection zone comprising a first capture zone comprising a first immobilized capture agent specific for a first target analyte, a second capture zone comprising a second immobilized capture agent specific for a second target analyte, and a third capture zone comprising a third immobilized capture agent specific for a third target analyte;

a first complex connected to the flow path in a first stage and configured to flow in the flow path to the detection zone in the presence of the fluid sample in a second stage, the first complex comprising a label, a first antibody or fragment thereof that specifically binds to the first target analyte, and the first target analyte;

a labeled second antibody or fragment thereof that specifically binds to the second target analyte, the labeled second antibody or fragment thereof being linked to the flow path in the first stage and configured to flow in the flow path to the detection zone in the presence of the fluid sample in the second stage; and

a labeled third antibody or fragment thereof that specifically binds to the third analyte of interest, the labeled third antibody or fragment thereof being linked to the flow path in the first phase and configured to flow in the flow path to the detection zone in the presence of the fluid sample in the second phase.

30. The assay test strip of claim 29, wherein the flow path is configured to receive a fluid sample including an unlabeled first target analyte, and wherein the first immobilized capture agent is configured to competitively bind the first complex and the unlabeled first target analyte in the fluid sample.

31. The assay test strip of claim 30, wherein the fluid sample further includes an unlabeled second target analyte at a concentration six orders of magnitude less than the concentration of the unlabeled first target analyte in the fluid sample.

32. The assay test strip of claim 30, wherein the fluid sample further includes an unlabeled third target analyte at a concentration six orders of magnitude less than the concentration of the unlabeled first target analyte in the fluid sample.

33. The assay test strip of claim 29, wherein the first complex flows to the first capture area and binds to the first immobilized capture agent at a third stage, and wherein the labeled second antibody or fragment thereof that binds to the second target analyte at a third stage flows to the second capture area and binds to the second immobilized capture agent.

34. The assay test strip of claim 33, wherein the first complex emits a first signal from the first capture area at a third stage, and wherein the labeled second antibody or fragment thereof that binds a second target analyte emits a second signal from the second capture area at the third stage.

35. The assay test strip of claim 34, wherein the flow path is configured to receive a fluid sample including an unlabeled first target analyte, and wherein the first signal emitted by the first capture region decreases as a concentration of the unlabeled first target analyte in the fluid sample increases.

36. The assay test strip of claim 35, wherein the fluid sample further includes an unlabeled second target analyte, and wherein the second signal emitted by the second capture region increases as the concentration of the unlabeled second target analyte in the fluid sample increases.

37. The assay test strip of claim 29, wherein the flow path is configured to receive a fluid sample including an unlabeled first target analyte, and wherein the first complex does not specifically bind the unlabeled first target analyte in a first stage or a second stage.

38. The assay test strip of claim 37, wherein in a second stage the first complex is configured to flow in the flow path with the unlabeled first target analyte to the first capture zone.

39. The assay test strip of claim 38, wherein the first complex is configured to compete with the unlabeled first target analyte for binding to the first immobilized capture agent in the first capture area at a third stage.

40. The assay test strip of claim 39, wherein a first optical signal emitted from a first complex bound to the first immobilized capture agent in the first capture zone decreases as the concentration of unlabeled first target analyte in the fluid sample increases.

41. The assay test strip of claim 29, wherein the flow path is configured to receive a fluid sample that includes or does not include a first target analyte, and wherein the first complex specifically binds all or substantially all of the first immobilized capture agent in the first capture zone in the second phase when the fluid sample does not include a first target analyte.

42. The assay test strip of claim 41, wherein when the fluid sample does not include a first target analyte, a first optical signal emitted by the first complex bound in the first capture zone is a maximum optical signal that the first capture zone of the assay test strip can emit.

43. The assay test strip of claim 42, wherein when the fluid sample includes a first target analyte, a first optical signal emitted by the first complex bound in the first capture zone is less than the maximum optical signal.

44. The assay test strip of claim 29, wherein the first immobilized capture agent comprises an antibody or fragment of an antibody that specifically binds the first analyte of interest.

45. The assay test strip of claim 29, wherein the first composite is integrated onto a surface of the assay test strip in a first stage.

46. The assay test strip of claim 29, wherein the first complex is integrated onto the surface of the assay test strip by spraying a solution comprising the first complex onto the surface of the assay test strip and drying the solution.

47. The assay test strip of claim 29, wherein the fluid sample is selected from the group consisting of a blood, plasma, urine, sweat, and saliva sample.

48. The assay test strip of claim 29, wherein the fluid sample is selected from the group consisting of whole blood, venous blood, capillary blood, serum, and plasma.

49. The assay test strip of claim 29, wherein the first target analyte comprises a C-reactive protein (CRP), the first complex comprises an anti-CRP antibody or fragment thereof that binds CRP, the second target analyte comprises TNF-related apoptosis-inducing ligand (TRAIL), and wherein the third target analyte comprises interferon gamma-induced protein 10 (IP-10).

50. A diagnostic test system, comprising:

the assay test strip of claim 29;

a reader comprising a light source and a detector; and

a data analyzer.

51. The diagnostic test system of claim 50, wherein the data analyzer outputs an indication of the absence of a first target analyte in the fluid sample when the reader detects that a first optical signal from the first capture zone of the assay test strip is the maximum optical signal of a dose response curve of the first capture zone of the test strip.

52. The diagnostic test system of claim 51, wherein the data analyzer outputs an indication of the presence of a low concentration of a first target analyte in the fluid sample when the reader detects an optical signal from the first capture area of the assay test strip that is within 1% of the maximum optical signal.

53. The diagnostic test system of claim 51, wherein the data analyzer outputs an indication of the presence of a low concentration of a first target analyte in the fluid sample when the reader detects an optical signal from the first capture area of the assay test strip that is within 5% of the maximum optical signal.

54. The diagnostic test system of claim 51, wherein the data analyzer outputs an indication of the presence of a low concentration of a first target analyte in the fluid sample when the reader detects an optical signal from the first capture area of the assay test strip that is within 10% of the maximum optical signal.

55. The diagnostic test system of claim 51, wherein the data analyzer outputs an indication of the presence of a high concentration of a first target analyte in the fluid sample when the reader detects an optical signal from the first capture area of the assay test strip that is 90% or less than 90% of the maximum optical signal.

56. The diagnostic test system of claim 51, wherein the data analyzer outputs an indication of the concentration of a first target analyte in the fluid sample when the reader detects that the optical signal from the first capture area of the assay test strip is below the maximum optical signal.

57. The diagnostic test system of claim 51, wherein the data analyzer outputs an indication of a concentration of a second target analyte in the fluid sample when the reader detects a second optical signal from the second capture zone of the assay test strip, wherein the indicated concentration of the second target analyte in the fluid sample is six orders of magnitude less than the indicated concentration of the first target analyte in the fluid sample.

58. The diagnostic test system of claim 51, wherein the data analyzer outputs an indication of a concentration of a third target analyte in the fluid sample when the reader detects a third optical signal from the third capture zone of the assay test strip, wherein the indicated concentration of the third target analyte in the fluid sample is six orders of magnitude less than the indicated concentration of the first target analyte in the fluid sample.

59. The diagnostic test system of claim 50, wherein the data analyzer outputs an indication that a second target analyte is not present in the fluid sample when the reader does not detect a second optical signal from the second capture zone of the assay test strip.

60. The diagnostic test system of claim 50, wherein the data analyzer outputs an indication that a third target analyte is not present in the fluid sample when the reader does not detect a third optical signal from the third capture zone of the assay test strip.

61. A method of determining the presence or concentration of each of a plurality of target analytes in a fluid sample, the method comprising:

applying the fluid sample to the assay test strip of claim 29 when the first complex, labeled second antibody or fragment thereof, and labeled third antibody or fragment thereof are each connected to a flow path at a first stage;

allowing a second analyte, if present in the fluid sample, to bind to the labeled second antibody or fragment thereof, thereby forming a second complex;

allowing a third analyte, if present in the fluid sample, to bind to the labeled third antibody or fragment thereof, thereby forming a third complex;

(ii) disengaging the first complex, the second complex (if formed), and the third complex (if formed) from the flow path;

flowing the fluid sample to the detection zone in a second stage;

binding the first complex to a first immobilized capture agent in a first capture zone, binding the second complex (if formed) to a second immobilized capture agent in a second capture zone, and binding the third complex (if formed) to a third immobilized capture agent in a third capture zone;

detecting a first signal from the first complex bound to the first immobilized capture agent in the first capture zone;

detecting a second signal from the second complex bound to the second immobilized capture agent in the second capture zone if the second complex is formed; and

detecting a third signal from the third complex bound to the third immobilized capture agent in the third capture zone if the third complex is formed.

62. The method of claim 61, wherein the first target analyte is present in the fluid sample at a concentration that is about six orders of magnitude greater than the concentration of the second target analyte present in the fluid sample.

63. The method of claim 61, wherein the first analyte of interest is present in the fluid sample at a concentration of between 1 and 999 μ l/ml and the second analyte of interest is present in the fluid sample at a concentration of between 1 and 999 pg/ml.

64. The method of claim 61, wherein the first target analyte is present in the fluid sample at a concentration at least one order of magnitude greater than a concentration of the second target analyte present in the fluid sample, the order of magnitude comprising one order of magnitude, two orders of magnitude, three orders of magnitude, four orders of magnitude, five orders of magnitude, six orders of magnitude, seven orders of magnitude, eight orders of magnitude, nine orders of magnitude, or ten orders of magnitude.

65. The method of claim 61, further comprising correlating the first signal with a concentration of the first target analyte present in the sample, and correlating the second signal with a concentration of the second target analyte present in the sample.

66. The method of claim 61, wherein the first signal detected by the first complex bound to the first immobilized capture agent in the first capture zone decreases as the concentration of the first analyte in the sample decreases, and wherein the second signal detected by the second complex bound to the second immobilized capture agent in the second capture zone increases as the concentration of the second target analyte in the sample increases.

Technical Field

The present disclosure relates generally to lateral flow assay devices, test systems, and methods. More particularly, the present disclosure relates to lateral flow assay devices that determine the presence and concentration of multiple analytes in a sample, including when one or more target analytes are present in high concentrations and one or more target analytes are present in low concentrations. When a single sample is applied to a single lateral flow assay in a single application, the precise concentration of each of the multiple analytes can be determined, including when the first target analyte is present in the single sample at one part per million of the concentration of the second target analyte in the single sample.

Background

Immunoassay systems comprising the lateral flow assays described herein provide reliable, inexpensive, portable, rapid, and simple diagnostic tests. Lateral flow assays can rapidly and accurately detect the presence or absence of a target analyte in a sample, and in some cases, can also quantify the target analyte in the sample. Advantageously, the lateral flow assay may be minimally invasive and may be used as a point-of-care testing system. Lateral flow assays have been developed to detect a variety of medical or environmental analytes. In a sandwich format lateral flow assay, a labeled antibody to a target analyte is deposited on the test strip in or near the sample receiving zone. The labeled antibody may include, for example, a detector molecule or "label" that binds to the antibody. When a sample is applied to the test strip, analyte present in the sample is bound by the labeled antibody, which flows along the test strip to the capture zone where the immobilized antibody to the analyte binds to the labeled antibody-analyte complex. The antibody immobilized on the capture line may be different from the labeled antibody deposited in or near the sample receiving zone. Detecting the captured complex and determining the presence of the analyte. In the absence of analyte, the labeled antibody flows along the test strip, passing through the capture zone. The absence of signal at the capture zone indicates the absence of analyte. Multiple assays can be developed to detect more than one analyte of interest present in a single sample applied to a lateral flow assay, but such assays have a number of disadvantages, including cross-reactivity between antibodies and analytes of interest; the inability to detect multiple target analytes applied to a single test strip using one optical reader in a single test event; and the inability to detect target analytes present at significantly different concentrations in a single sample. Typically, a sample having a high concentration of analyte must first be diluted to test for the presence or concentration of the high concentration of analyte. This dilution further reduces the concentration of any target analyte present in the sample at low concentrations, making low concentrations undetectable. To date, multiplexed lateral flow assays have not been suitable for determining the quantity and presence of multiple analytes in a sample, where one or more analytes are present in high concentrations and one or more analytes are present in low concentrations.

Disclosure of Invention

Accordingly, it is an aspect of the present disclosure to provide an improved lateral flow assay for detecting the presence and concentration of multiple analytes of interest in a sample when a first analyte is present in the sample at a high concentration and a second, different analyte is present in the sample at a low concentration (including, but not limited to, parts per million concentrations of high concentrations).

In one embodiment of the present disclosure, a method of detecting a first target analyte and a second target analyte present at different concentrations in a sample is provided. The method includes providing a lateral flow assay comprising a first complex linked to a flow path of the lateral flow assay, the first complex comprising a label, an antibody or fragment thereof that specifically binds to a first analyte, and the first analyte. The lateral flow assay further comprises a labeled second antibody or fragment thereof coupled to the flow path and configured to specifically bind the second analyte. The lateral flow assay further comprises a first capture zone downstream of the first complex, the first capture zone comprising a first immobilized capture agent specific for the first analyte. The lateral flow assay further comprises a second capture zone downstream of the labeled second antibody or fragment thereof and comprising a second immobilized capture agent specific for the second analyte. The method further comprises applying the sample to the first complex and a labeled second antibody or fragment thereof; and binding the second analyte to a labeled second antibody or fragment thereof to form a second complex. The method further comprises flowing the fluid sample and the first complex to the first capture zone, wherein the first analyte in the fluid sample and the first complex compete for binding to the first immobilized capture agent in the first capture zone; and flowing the second complex in the flow path to the second capture zone and allowing the second complex to bind to the second immobilized capture agent in the second capture zone. The method also includes detecting a first signal from a first complex bound to the first immobilized capture agent in the first capture zone and a second signal from a second complex bound to the second immobilized capture agent in the second capture zone.

In another embodiment of the present disclosure, a lateral flow assay configured to detect a first target analyte and a second target analyte present at different concentrations in a fluid sample is provided. The lateral flow assay comprises a first complex coupled to a flow path of the lateral flow assay, the first complex comprising a label, an antibody, or a fragment thereof that specifically binds to a first analyte, and the first analyte; a labeled second antibody or fragment thereof coupled to the flow path and configured to specifically bind to a second analyte; a first capture zone downstream of the first complex, the first capture zone comprising a first immobilized capture agent specific for a first analyte; and a second capture zone downstream of the labeled second antibody or fragment thereof and comprising a second immobilized capture agent specific for the second analyte.

In yet another embodiment of the present disclosure, an assay test strip is provided. The assay test strip includes a flow path configured to receive a fluid sample; a sample receiving zone connected to the flow path; and a detection zone connected to the flow path downstream of the receiving zone. The detection zone includes a first capture zone, a second capture zone, and a third capture zone. The first capture zone includes a first immobilized capture agent specific for a first target analyte, the second capture zone includes a second immobilized capture agent specific for a second target analyte, and the third capture zone includes a third immobilized capture agent specific for a third target analyte. The assay test strip also includes a first complex connected to the flow path in the first stage and configured to flow in the flow path to the detection zone in the presence of the fluid sample in the second stage. The first complex includes a label, a first antibody or fragment thereof that specifically binds to a first analyte of interest, and the first analyte of interest. The assay test strip further includes a labeled second antibody or fragment thereof that specifically binds a second target analyte, the labeled second antibody or fragment thereof being coupled to the flow path in the first stage and configured to flow in the flow path to the detection zone in the presence of the fluid sample in the second stage. The assay test strip also includes a labeled third antibody or fragment thereof that specifically binds a third target analyte, the labeled third antibody or fragment thereof being connected to the flow path in the first stage and configured to flow in the flow path to the detection zone in the presence of the fluid sample in the second stage.

In still further embodiments of the present disclosure, a diagnostic test system is provided. The diagnostic test system includes an assay test strip as described above; a reader comprising a light source and a detector, and a data analyzer.

In another embodiment of the present disclosure, a method of determining the presence or concentration of each of a plurality of target analytes in a fluid sample is provided. The method includes applying a fluid sample to the above-described assay test strip while the first complex, the labeled second antibody or fragment thereof, and the labeled third antibody or fragment thereof are each connected to the flow path in the first stage. The method further comprises binding a second analyte (if present in the fluid sample) to a labeled second antibody or fragment thereof, thereby forming a second complex; binding the third analyte (if present in the fluid sample) to the labeled third antibody or fragment thereof, thereby forming a third complex; (ii) detaching the first composite, the second composite (if formed), and the third composite (if formed) from the fluidic pathway; flowing the fluid sample to a detection zone in a second phase; binding the first complex to a first immobilised capture reagent in a first capture zone, binding the second complex (if formed) to a second immobilised capture reagent in a second capture zone, and binding the third complex (if formed) to a third immobilised capture reagent in a third capture zone; detecting a first signal from the first complex bound to the first immobilized capture agent in the first capture zone; detecting a second signal from the second complex bound to the second immobilized capture agent in the second capture zone if the second complex is formed; and detecting a third signal from the third complex bound to the third immobilized capture agent in the third capture zone if the third complex is formed.

Drawings

Fig. 1A and 1B illustrate an example lateral flow assay according to the present disclosure before and after application of a fluid sample at a sample receiving zone, wherein the fluid sample includes a first target analyte, a second target analyte, and a third target analyte.

Fig. 2A and 2B illustrate an example lateral flow assay according to the present disclosure before and after application of a fluid sample at a sample-receiving zone, wherein the fluid sample does not include any target analyte.

Fig. 3A and 3B illustrate an example lateral flow assay according to the present disclosure before and after application of a fluid sample at a sample-receiving zone, where the fluid sample includes a first target analyte but does not include a second or third target analyte.

Fig. 4A and 4B illustrate an example lateral flow assay according to the present disclosure before and after application of a fluid sample at a sample-receiving zone, where the fluid sample includes a second target analyte but does not include the first or third target analytes.

Fig. 5A and 5B illustrate an example lateral flow assay according to the present disclosure before and after application of a fluid sample at a sample-receiving zone, where the fluid sample includes a third target analyte but does not include the first or second target analytes.

Fig. 6A and 6B are cross-flow assays according to examples of the present disclosure before and after application of a fluid sample to a sample receiving zone, where the fluid sample includes first and third target analytes but does not include a second target analyte.

Fig. 7A illustrates an example dose response curve for an example lateral flow assay, such as that illustrated in fig. 3A and 3B, wherein the fluid sample comprises only C-reactive protein (CRP) at a concentration of up to 150 μ g/mL, and wherein the fluid sample does not comprise any additional analyte of interest, such as TNF-related apoptosis-inducing ligand (TRAIL) or interferon gamma-induced protein 10 (IP-10).

Fig. 7B illustrates an example dose response curve for an example lateral flow assay, such as that illustrated in fig. 4A and 4B, wherein the fluid sample includes only IP-10 at a concentration of up to 1000pg/mL, and wherein the fluid sample does not include any additional analyte of interest, such as TRAIL or CRP.

Fig. 7C illustrates an example dose response curve for an example lateral flow assay, such as that illustrated in fig. 5A and 5B, wherein the fluid sample comprises only TRAIL at a concentration of up to 500pg/mL, and wherein the fluid sample does not comprise any additional analyte of interest, such as IP-10 or CRP.

Fig. 8 illustrates an example lateral flow assay device according to this disclosure that includes a sample receiving zone and a detection zone. The detection zone may include an indication of the presence and/or concentration of multiple analytes in the fluid sample, such as, but not limited to, CRP, IP-10, and TRAIL, including when one or more analytes of interest are present at high concentrations and one or more analytes of interest are present at low concentrations.

Detailed Description

The devices, systems, and methods described herein accurately determine the quantity or presence of multiple target analytes in a sample. Lateral flow devices, test systems, and methods according to the present disclosure accurately determine the presence or quantity of multiple target analytes in situations where one or more target analytes are present in a sample at an elevated or high concentration and one or more target analytes are present in a sample at a low concentration. Advantageously, the lateral flow devices, test systems, and methods described herein determine the presence or amount of target analytes present at significantly different concentrations in a single sample after the single sample is applied to one lateral flow assay (such as a single test strip) in a single test event. Thus, the lateral flow assays described herein are capable of detecting multiple analytes in a single sample simultaneously, even when the analytes are present in significantly different concentration ranges.

The lateral flow assays described herein may use a combination of binding assays on a single test strip, including an assay for detecting one or more analytes present at high concentrations in combination with an assay for detecting one or more analytes present at low concentrations. A single test strip for a lateral flow assay described herein may include a detection zone having a separate capture zone specific for each analyte of interest. For example, the sample may include three target analytes: a first target analyte, a second target analyte, and a third target analyte. The detection zone for the lateral flow assay will therefore comprise three capture zones: a first capture zone specific for a first target analyte, a second capture zone specific for a second target analyte, and a third capture zone specific for a third target analyte.

In this non-limiting example, the first analyte of interest can be present in the sample at a high concentration, such as, but not limited to, a range of 1-999 μ g/ml. The lateral flow assays described herein can produce a signal of maximum intensity at the first capture zone when the concentration of the first target analyte in the sample is zero. Increasing the concentration of the first target analyte reduces the signal from a maximum intensity signal to a reduced intensity signal, which may be correlated to the concentration of the first target analyte. In this example, the second analyte of interest and the third analyte of interest may be present in the sample at low concentrations, such as, but not limited to, in the range of 1-999 pg/ml. The lateral flow assays described herein can produce signal intensities at the second capture zone and the third capture zone, where increased signal intensities correlate with increased concentrations of the second target analyte and the third target analyte, respectively. Thus, a lateral flow assay according to the present disclosure can detect both high and low concentrations of an analyte using a single assay, such as a single test strip.

A lateral flow assay according to the present disclosure can measure the presence and concentration of multiple target analytes present at significantly different concentrations in a single undiluted sample applied to a single lateral flow assay in a single test event. The ability to measure the presence and concentration of multiple target analytes at very different concentrations (including concentrations that differ by six orders of magnitude, or by a million-fold) without diluting the sample provides significant advantages. For example, embodiments of the lateral flow assay described herein can measure analytes present in whole blood, venous blood, capillary blood, serum, and plasma samples that have not been diluted or pretreated prior to application to a lateral flow assay (such as a single lateral flow assay test strip).

Advantageously, the implementation of a lateral flow assay can simultaneously detect low concentrations of an analyte present in the same sample as high concentrations of the analyte, even if the high concentration of the analyte has a large dynamic range (including, but not limited to, CRP, which may be present in the sample with a larger dynamic range). In addition, the ability to simultaneously and accurately detect the concentration of multiple target analytes present at significantly different concentrations (on the order of parts per million) in a single sample has significant diagnostic benefits. In one non-limiting embodiment of the lateral flow assay of the present disclosure, the measurement of the optical signal from a single test strip can be correlated with the presence or absence of a viral infection, a bacterial infection, or no infection in the patient.

In the context of optical signals generated by reflective labels (such as, but not limited to, gold nanoparticle labels), signals generated by assays according to the present disclosure are described herein. Although embodiments of the present disclosure are described herein with reference to "optical" signals, it is to be understood that the assays described herein may use any suitable material for the label to produce a detectable signal, including but not limited to fluorescent latex bead labels that produce a fluorescent signal and magnetic nanoparticle labels that produce a signal indicative of a change in a magnetic field associated with the assay.

According to the present disclosure, a lateral flow assay device includes a labeled antibody designed to detect a high concentration of an analyte in a sample in combination with a labeled antibody designed to detect a low concentration of an analyte in the same sample. For example, the sample may include a high concentration of a first target analyte, a low concentration of a second target analyte, and a low concentration of a third target analyte. To detect a high concentration of a first analyte of interest, a first complex is first integrated onto the surface of a receiving zone or label zone of a lateral flow assay test strip, e.g., onto a conjugate pad. The first complex includes a label, a first antibody that specifically binds to a first analyte of interest, and the first analyte of interest. The first complex becomes unbound to the label region when the fluid sample, which may include the first analyte of interest, is applied to the test strip and travels with the fluid sample to the detection region of the test strip. The detection zone includes a capture zone specific for each target analyte and thus includes a first capture zone to capture a first target analyte, a second capture zone to capture a second target analyte, and a third capture zone to capture a third target analyte. The first complex and the first analyte of interest in the sample (when present) bind to the first capture agent in the first capture zone. The first capture agent binds only to the first complex when the first analyte of interest (which would otherwise compete with the first complex) is not present in the sample. Thus, when the first target analyte is not present in the sample, a first signal having a maximum intensity is generated at the first capture zone. When the first analyte of interest is present in the sample at a low concentration, the first complex competes with a relatively low amount of the first analyte for binding to the first capture agent, resulting in a first signal that is the same as, or substantially equal to (within a limited range of variance from) the first signal having the greatest intensity. When the first analyte of interest is present in the sample at a high concentration, the first complex competes with a relatively high amount of the first analyte for binding to the first capture agent, resulting in a first signal that is less than the signal having the greatest intensity.

To detect a second target analyte (which in this non-limiting example is present in the sample at a low concentration), a labeled second antibody that specifically binds to the second target analyte is first incorporated onto the surface of the lateral flow assay test strip receiving area or label area, for example, onto a conjugate pad. The labeled second antibody becomes unbound to the label region and binds to the second target analyte to form a second complex when the fluid sample is applied to the test strip. The second complex travels with the fluid sample to a detection zone of the test strip. The second complex is bound in a second capture zone to a second capture agent specific for a second target analyte. As a result, a second signal is generated at the second capture area when a second target analyte is present in the sample. When the second analyte of interest is not present in the sample (or is present at a level below detectable), no second complex is formed (or less than a detectable amount of the second complex is formed), and thus no second complex is captured at the second capture zone (or no detectable amount of the second complex is captured at the second capture zone). In this case, the labeled second antibody travels with the fluid sample to the detection zone of the test strip, but it does not bind to the second capture reagent at the second capture zone. As a result, no second signal is detected at the second capture area. The signal intensity of the second signal correlates to the concentration of the second target analyte, wherein an increased signal intensity correlates to an increased concentration of the second target analyte in the sample.

Similarly, to detect a third analyte of interest (which in this non-limiting example is present in the sample at a low concentration), a labeled third antibody that specifically binds to the third analyte of interest is first integrated onto the surface of the lateral flow assay test strip receiving or label zone, e.g., onto the conjugate pad. The labeled third antibody becomes unbound to the label region when the fluid sample is applied to the test strip and binds to a third analyte of interest to form a third complex. The third complex travels with the fluid sample to a detection zone of the test strip. The third complex is bound in a third capture zone to a third capture agent specific for a third target analyte. As a result, a third signal is generated at the third capture area when a third target analyte is present in the sample. When the third analyte of interest is not present in the sample (or is present at a level below detectable), no third complex is formed (or less than a detectable amount of the third complex is formed), and thus no third complex is captured at the third capture zone (or no detectable amount of the third complex is captured at the third capture zone). In this case, the labeled third antibody travels with the fluid sample to the detection zone of the test strip, but it does not bind to the third capture agent at the third capture zone. As a result, no third signal is detected at the third capture area. The signal intensity of the third signal correlates to the concentration of the third target analyte, wherein an increased signal intensity correlates to an increased concentration of the third target analyte in the sample.

The above description is intended to illustrate that a fluid sample may include a first target analyte present in a high concentration, a second target analyte present in a low concentration, and a third target analyte present in a low concentration. Those skilled in the art will recognize that the examples are intended to be exemplary and that various modifications and variations can be employed with respect to the lateral flow assays described herein. For example, the fluid sample may include only two analytes of interest, wherein a first analyte is present in a high concentration and wherein a second analyte is present in a low concentration. Alternatively, the fluid sample may include three target analytes, wherein a first target analyte is present in a high concentration, a second target analyte is present in a high concentration, and a third target analyte is present in a low concentration. Further, the fluid sample may include more than three (such as four, five, six, seven, eight, nine, and ten) target analytes with various iterations (iterations) of many analytes present at high and low concentrations. In each of the various iterations, the lateral flow assay is designed as described above to detect both the amount and presence of high concentration analytes and the amount and presence of low concentration analytes simultaneously and on a single lateral flow assay device.

Those skilled in the art will recognize that high and low concentrations are relative terms and that the following non-limiting examples are intended to be illustrative and not limiting of the disclosure. In some non-limiting implementations described below, a first "low concentration" analyte is present in a sample at one part per million of the concentration at which a second, different "high concentration" analyte is present in the same sample. Lateral flow assays according to the present disclosure can measure the presence and concentration of analytes present at different magnitudes of concentration, including but not limited to the presence of a first target analyte at one, two, three, four, five, six, seven, eight, nine, and ten magnitudes greater than the concentration of a second, different target analyte.

Without being bound by any particular theory, the operation of a first complex (which includes a label, a first antibody that specifically binds a first analyte of interest, and a first analyte of interest) together with a second labeled antibody that specifically binds a second analyte of interest, both integrated in a label zone of a single lateral flow assay, for the simultaneous detection and quantification of a high concentration of analyte and a low concentration of analyte, will now be described. Without being bound by any particular theory, the first complex is used to mask the portion of the conventional sandwich lateral flow assay dose response curve where the signal increases (when the first analyte concentration is low), thereby generating a first dose response curve at the first capture zone that begins with a maximum intensity when the concentration of the first target analyte is zero and then remains relatively constant (the first analyte is at a low concentration) or decreases (the first analyte is at a high concentration). A second (or additional) labeled antibody that specifically binds a second analyte of interest produces a second dose response curve at the second capture zone that produces an increased signal intensity as the concentration of the second analyte increases. The lateral flow assay of the present disclosure addresses the deficiencies associated with measuring multiple target analytes in a sample, particularly where one or more target analytes are present in high concentrations and one or more target analytes are present in low concentrations.

In some cases, for example, a fluid sample may comprise a plurality of target analytes, wherein one or more target analytes are present in high concentrations and one or more target analytes are present in low concentrations. In particular, the one or more target analytes may be present in the sample in an amount that is millions of times greater than the amount of the one or more target analytes present in the low concentration. Previously, to address this problem, two or more separate tests were required to detect analytes present in significantly different concentrations in a fluid sample. For example, to detect high concentrations of an analyte, a sample may be diluted to reduce the high concentration of the analyte in the sample to a detectable concentration. Sample dilution requires additional physical steps to dilute the sample. In addition, dilution requires additional steps to calculate the amount of analyte, resulting in a more complex algorithm, which may affect the accuracy of the measured amount of analyte in the sample. In addition, dilution of the sample eliminates the ability to detect analytes present at low concentrations because the diluted sample causes the concentration of analytes present at low concentrations to fall below the detectable range. Thus, a single sample with both high and low concentrations of analyte may be diluted to determine the concentration of the high concentration analyte, but this same sample is not suitable for determining the concentration of the low concentration analyte in a conventional multiplex assay.

For detection of low concentrations of analyte, a sandwich lateral flow assay may be used. Conventional sandwich lateral flow assays are not suitable for, and in some cases cannot accurately determine, the amount of high concentrations of analytes. Thus, the detection of both high and low concentrations of analyte present in a single sample has previously required the application of the sample to multiple detection assays, each specifically designed to detect the presence of a particular target analyte within a particular dynamic range of that target analyte.

In contrast, the lateral flow assay described herein is capable of determining the presence and/or amount of multiple analytes in a fluid sample in a single test (such as a single application of a fluid sample to a single lateral flow assay test strip), wherein one or more target analytes are present in a high concentration in the fluid sample and one or more target analytes are present in a low concentration in the fluid sample.

The lateral flow assay described herein includes further advantageous features. For example, signals generated when the first analyte is at a high concentration are readily detected (e.g., their intensity is within a well-spaced optical signal range that is typically discernable by conventional readers), they do not overlap on the dose response curve with signals generated by zero or low concentrations of the first analyte, and they can be used to calculate highly accurate concentration readings at high and even very high concentrations. In some advantageous implementations, the intensity level of the signal generated when the first analyte is present at a high concentration does not overlap with the intensity level of the signal generated when the first analyte is present at a low concentration.

Embodiments of the lateral flow assay described herein are particularly advantageous in diagnostic tests for a plurality of target analytes, where the relative concentrations of the plurality of target analytes are indicative of a disease state. A diagnosis of a particular disease state can be positively determined when one target analyte is present at a higher concentration than in the normal or healthy state, but the other target analyte is unchanged from the normal or healthy state.

Examples of analytes that can be detected and measured by the lateral flow assay devices, test systems, and methods of the present disclosure include, but are not limited to, the following proteins: TRAIL, CRP, IP-10, PCT and MX 1. Implementations of the disclosure can measure the soluble form and/or membrane form of a TRAIL protein. In one embodiment, only the soluble form of TRAIL is measured.

Various aspects of the devices, test systems, and methods are described more fully hereinafter with reference to the accompanying drawings. However, the present disclosure may be embodied in many different forms. Based on the teachings herein one skilled in the art should appreciate that the scope of the present disclosure is intended to cover any aspect of the devices, test systems, and methods disclosed herein, whether implemented independently of or in combination with any other aspect of the present disclosure. For example, an apparatus may be implemented or a method may be practiced using any number of the aspects set forth herein.

Although specific aspects are described herein, many variations and permutations of these aspects fall within the scope of the present disclosure. Although some benefits and advantages are mentioned, the scope of the present disclosure is not intended to be limited to the particular benefits, uses, or objectives. Rather, aspects of the present disclosure are intended to be broadly applicable to different detection techniques and apparatus configurations, some of which are illustrated by way of example in the accompanying drawings and the following description. The detailed description and drawings are merely illustrative of the disclosure rather than limiting, the scope of the disclosure being defined by the appended claims and equivalents thereof.

The lateral flow device described herein is an analytical device used in lateral flow chromatography. A lateral flow assay is an assay that can be performed on the lateral flow device described herein. The lateral flow device may be implemented on a test strip, but other forms may also be suitable. In a test strip format, a test fluid sample suspected of containing an analyte flows (e.g., by capillary action) through the test strip. The test strip may be made of a bibulous material such as paper, nitrocellulose, and cellulose. A fluid sample is received in a sample reservoir. The fluid sample can flow along the test strip to the capture zone, where the analyte (if present) interacts with the capture agent to indicate the presence, absence, and/or quantity of the analyte. The capture agent may comprise an antibody immobilized in a capture zone.

The lateral flow assay may be performed in a sandwich format. The sandwich and assay described herein will be described in the context of a reflective label that generates an optical signal (such as a gold nanoparticle label), but it should be understood that the assay may include a latex bead label configured to generate a fluorescent signal, a magnetic nanoparticle label configured to generate a magnetic signal, or any other label configured to generate a detectable signal. Sandwich-type lateral flow assays include labeled antibodies deposited at a sample reservoir on a solid substrate. After applying the sample to the sample reservoir, the labeled antibody dissolves in the sample, after which the antibody recognizes and binds to a first epitope on the analyte in the sample, forming a label-antibody-analyte complex. The complex flows along a liquid front (front) from the sample reservoir through the solid substrate to a capture zone (sometimes referred to as a "test line") where an immobilized antibody (sometimes referred to as a "capture agent") is disposed. In some cases where the analyte is a multimer or contains multiple identical epitopes on the same monomer, the labeled antibody deposited at the same sample reservoir may be the same as the antibody immobilized at the capture region. The immobilized antibody recognizes and binds to an epitope on the analyte, thereby capturing the label-antibody-analyte complex at the capture zone. The presence of the labeled antibody at the capture zone provides a detectable optical signal at the capture zone. In one non-limiting embodiment, gold nanoparticles are used to label the antibodies because they are relatively inexpensive, stable, and readily provide observable color based on the surface plasmon resonance properties of the gold nanoparticles. In some cases, the signal provides qualitative information, such as whether an analyte is present in the sample. In some cases, the signal provides quantitative information, such as a measure of the amount of analyte in the sample.

The lateral flow assay may provide qualitative information, such as information regarding the presence or absence of a target analyte in a sample. For example, detection of any measurable optical signal at the capture zone may indicate the presence (in some unknown amount) of the target analyte in the sample. The absence of any measurable optical signal at the capture zone may indicate that the target analyte is not present in the sample or below the limit of detection. For example, if the sample does not contain any target analyte, the sample will still allow the labeled reagent to dissolve and the labeled reagent will still flow to the capture zone. However, the labelled reagent will not bind to the capture reagent at the capture zone. Instead, it will flow through the capture zone, and in some cases, to the optional absorption zone, via the control line (if present). Some labeled reagents will bind to the control agent deposited on the control line and emit a detectable optical signal. In these cases, the absence of a measurable optical signal emitted by the capture zone indicates the absence of the target analyte in the sample, while the presence of a measurable optical signal emitted by the control line indicates the sample traveling from the sample-receiving zone, through the capture zone, and to the capture line, as expected during normal operation of the lateral flow assay.

Some lateral flow devices can provide quantitative information, such as a measure of the amount of a target analyte in a sample. In particular, lateral flow assays can provide reliable quantitation of analytes when present in low concentrations. The quantitative measurement obtained by the lateral flow device may be the concentration of analyte present in a given volume of sample, obtained using a dose response curve that relates the intensity of the signal detected at the capture zone to the concentration of analyte in the sample. Example signals include optical signals, fluorescent signals, and magnetic signals. For sandwich-type lateral flow assays, if the sample does not contain any analyte of interest, the concentration of analyte in the sample is zero and no analyte binds to the labeled reagent to form a label-antibody-analyte complex. In this case, there is no complex that flows to the capture zone and binds to the capture antibody. Thus, no detectable optical signal is observed at the capture zone, and the signal amplitude is zero.

A signal is detected when the concentration of the analyte in the sample increases with increasing concentration of the analyte in the sample. This occurs because as the concentration of analyte increases, the formation of the label-antibody-analyte complex increases. The capture agent immobilized at the capture zone binds to the increased number of complexes flowing to the capture zone, resulting in an increase in the signal detected at the capture zone. Such assays provide reliable quantitation of analytes when present at low concentrations.

However, the above-described assay, which is suitable for quantifying a target analyte present at a low concentration, is not suitable for quantifying a target analyte present at a high concentration. In such cases, the concentration of the analyte may exceed the amount of labeled reagent available to bind the analyte, such that there is an excess of analyte. In this case, excess analyte bound by the unlabeled reagent will compete with the label-antibody-analyte complex for binding to the capture agent in the capture zone. The capture agent in the capture zone will bind to unlabeled analyte (in other words, analyte that is not bound to the labeled reagent) and label-antibody-analyte complexes. However, unlabeled analyte bound to the capture agent does not emit a detectable signal. As the concentration of analyte in the sample increases, the amount of unlabeled analyte bound to the capture agent (instead of the label-antibody-analyte complex that emits a detectable signal) also increases. As more and more unlabeled analyte binds to the capture agent instead of the label-antibody-analyte complex, the signal detected at the capture zone decreases.

This phenomenon, in which the detected signal initially increases at low concentrations and the detected signal decreases at high concentrations, is referred to as the "hook effect". As the concentration of analyte increases, more analyte binds to the labeled reagent, resulting in an increase in signal intensity. At the saturation concentration, the labeled reagent is saturated with the analyte in the sample (e.g., the available amount of labeled reagent has completely or nearly completely bound to the analyte from the sample), and the detected signal has reached a maximum signal intensity. As the concentration of analyte in the sample continues to increase beyond the maximum signal intensity, the presence of signal detected decreases as excess analyte above the saturation point of the labeled reagent competes with the labeled reagent analyte for binding to the capture reagent.

The hook effect, also referred to as the "prozone effect," adversely affects lateral flow assays, particularly where the analyte of interest is present in the sample at an elevated concentration. The hook effect can lead to inaccurate test results. For example, the hook effect can lead to false negatives or inaccurately low results. In particular, inaccurate results occur when the sample contains elevated levels of analyte that exceed the concentration of the labeled reagent deposited on the test strip. In this scenario, when the sample is placed on the test strip, the labeled reagent is saturated, and not all of the analyte is labeled. The unlabeled analyte flows through the analyte and competes strongly for binding to the labeled complex at the capture zone, thereby reducing the detectable signal. Thus, since a single detected signal corresponds to both a low and a high concentration, the device (or an operator of the device) cannot distinguish whether the optical signal corresponds to a low or a high concentration. If the analyte level is sufficiently high, the analyte competes completely with the labeled complex and no signal is observed at the capture zone, resulting in a false negative test result.

Example lateral flow device for accurately quantifying multiple analytes present in both high and low concentrations in a single sample Device for placing

The lateral flow assay, test system, and methods described herein address these and other shortcomings of multiple sandwich-type lateral flow assays. Fig. 1A-6B illustrate example lateral flow assays that can accurately measure the amount of multiple target analytes, where one or more target analytes are present in high concentrations and one or more target analytes are present in low concentrations in a single sample. Fig. 7A-7C provide graphs illustrating example dose response curves for optical signals measured by the lateral flow assays described herein and, in particular, the relationship between the amplitude of the optical signal detected at the capture zone (measured along the y-axis) and the concentration of analyte in the sample applied to the assay (measured along the x-axis). It should be understood that while assays according to the present disclosure are described in the context of reflective labels that generate an optical signal, assays according to the present disclosure may include labels of any suitable material configured to generate a fluorescent signal, a magnetic signal, or any other detectable signal.

The lateral flow assay devices, systems, and methods described herein are capable of detecting the presence or determining the concentration of multiple analytes in a sample, where one or more analytes are present in high concentrations and one or more analytes are present in low concentrations. In some embodiments, a first analyte of interest present in a sample at a high concentration may be present in an amount that is 1 million, 9 million, 8 million, 7 million, 6 million, 5 million, 4 million, 3 million, 2 million, 1 million, 500,000, 100,000, 50,000, 10,000, 5,000, 1,000, 500, 100, or 10 times greater than the amount of a second, different analyte also present in the sample but at a low, very low, or very low concentration. In some cases, the second target analyte is present in a trace amount compared to the first target analyte in a given volume of the fluid sample. For example, a high concentration of analyte may be present in an amount of 10 to 100 μ g/mL (10,000,000 to 100,000,000pg/mL), while a low concentration of analyte may be present in an amount of 10 to 100 pg/mL.

The example lateral flow assay 101 illustrated in fig. 1A-6B includes a test strip having a sample receiving zone 110, a label zone 120, and a detection zone 130, wherein the detection zone includes a first capture zone 135, a second capture zone 133, and a third capture zone 131. Fig. 1A and 1B illustrate lateral flow device 101 before and after fluid sample 111 has been applied to sample reservoir 110, where the fluid sample includes first target analyte 112, second target analyte 113, and third target analyte 114. In the illustrated example, the marker region 120 is downstream of the sample receiving region 110 in the direction of sample flow within the test strip. In some cases, the sample receiving zone 110 is located within the label zone 120 and/or is co-located with the label zone 120. A first capture agent 136 is immobilized in the first capture area 135, a second capture agent 134 is immobilized in the second capture area 133, and a third capture agent 132 is immobilized in the third capture area 131.

In the practice of the present disclosure, the first complex 121 is integrated on the label region 120. The first complex 121 includes the label 124, a first antibody that specifically binds the first analyte of interest 112, and the analyte of interest 112. A second labeled antibody 123 is integrated on the label region 120. The second labeled antibody 123 includes a label 124 and a second antibody that specifically binds to a second target analyte 113. A third labeled antibody 122 is integrated onto the label region 120. The third labeled antibody 122 includes a label 124 and a third antibody that specifically binds the third target analyte 114. As illustrated in fig. 1A-6B, the label 124 is the same for each of the first complex 121, the second labeled antibody 123, and the third labeled antibody 122. It will be appreciated that the label 124 may be identical for each of the first complex 121, the second labeled antibody 123, and the third labeled antibody 122. Alternatively, the label may be different for each of the first complex 121, the second labeled antibody 123, and the third labeled antibody 122. Thus, the labels may provide the same or different optical signals for each of a plurality of target analytes. The label may be a reflective label that generates an optical signal, a latex bead label configured to generate a fluorescent signal, a magnetic nanoparticle configured to generate a magnetic signal, or any other label configured to generate a detectable signal.

For example, the label may be any substance, compound, or particle that can be detected, such as by visual, fluorescent, radiative, or instrumental mechanisms. The marker may be, for example, a pigment or ink produced as a colorant, such as Brilliant Blue, 132Fast Red 2R, and 4230Malachite Blue Lake. The label may be a particulate label such as a blue latex bead, gold nanoparticle, colored latex bead, magnetic particle, carbon nanoparticle, selenium nanoparticle, silver nanoparticle, quantum dot, upconversion phosphor, organic fluorophore, textile dye, enzyme, or liposome.

In some cases, the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 are formed and applied to a test strip, which is then used by an operator. For example, the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 can be integrated in the label region 120 during test strip manufacturing. In another example, the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 are integrated in the label region 120 after manufacture and prior to applying the fluid sample 111 to the test strip. The first complex 121, the second labeled antibody 123, and the third labeled antibody 122 can be integrated into the test strip in a variety of ways, as discussed in detail below.

Thus, in embodiments of the lateral flow device of the present disclosure, the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 are formed and integrated on the test strip before any fluid sample 111 has been applied to the lateral flow device 101. In one non-limiting embodiment, the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 are formed and integrated onto the conjugate pad of the test strip prior to any fluid sample 111 being applied to the lateral flow device 101. Furthermore, in embodiments of the lateral flow device of the present disclosure, the analyte in the first complex 121 is not an analyte from the fluid sample 111.

To conduct a test in which test strip 101 is applied, sample 111 having first, second, and third target analytes 112, 113, and 114 is deposited on sample-receiving zone 110, as shown in fig. 1A and 1B. In the illustrated embodiment where the label zone 120 is located downstream of the sample receiving region 110, the first 112, second 113 and third 114 target analytes in the sample 111 flow into the label zone 120 and contact the integrated first 121, second 123 and third 122 complexes. The sample 111 dissolves the first complex 121, the second labeled antibody 123 and the third labeled antibody 122. In one non-limiting embodiment, the sample 111 dissolves the first complex 121, the second labeled antibody 123, and the third labeled antibody 122. The bonds holding the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 to the surface of the test strip in the label region 120 are released so that the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 are no longer integrated onto the surface of the test strip. The second labeled antibody 123 binds to the second target analyte 113 in the sample to form a second complex, and the third labeled antibody 122 binds to the third target analyte 114 in the sample to form a third complex.

The first complex 121, the second complex, and the third complex along with the first analyte 112 (unbound) in the sample 111 migrate along the fluid front to the detection zone 130. The first capture agent 136 at the first capture zone 135 binds to the first complex 121 and the first analyte 112 in the sample 111. The second capture agent 134 at the second capture zone 133 binds to the second complex and the third capture agent 132 at the third capture zone 131 binds to the third complex.

In the practice of the present disclosure, depending on the amount of the first analyte 112 in the sample 111, the first complex 121 and the first analyte 112 compete with each other for binding to the first capture agent 136 in the first capture zone 135. A first detectable signal is detected at the first capture zone 135, wherein in the presence of the first target analyte 112 in the sample, the first detectable signal decreases from a signal of maximum intensity because the first target analyte 112 competes with the first complex 121 for binding to the first capture agent 136 at the first capture zone. In contrast, a second detectable signal is detected at the second capture area 133 and increases in intensity as the concentration of the second target analyte 113 in the sample increases, as the second target analyte 113 forms a second complex that emits a detectable signal at the second capture area 133. Similarly, a third detectable signal is detected at the third capture area 131, and the intensity increases with increasing concentration of the third target analyte 114 in the sample, as the third target analyte 114 forms a third complex that emits a detectable signal at the third capture area 131.

Thus, a lateral flow device according to the present disclosure comprises: a first complex comprising a label, a first antibody that specifically binds to a first analyte of interest, and the first analyte of interest; a second labeled antibody that specifically binds a second analyte of interest; and a third labeled antibody that specifically binds a third target analyte, each of which binds to a label region of the lateral flow device at a first stage (e.g., prior to application of the fluid sample to the lateral flow device) and then migrates through the test strip at a second, subsequent stage (e.g., upon application of the fluid sample to the sample receiving region). In a third stage (e.g., after the fluid sample flows to the detection zone), the first complex can bind to the first capture agent in the first capture zone, the second complex can bind to the second capture agent in the second capture zone, and the third complex can bind to the third capture agent in the third capture zone. Thus, the first complex, second labeled antibody, and third labeled antibody described herein can be initially positioned in a first region (such as a label region) of a lateral flow device, then migrate with the fluid (upon contact with the fluid) to other regions of the lateral flow device downstream of the first region, and then bind with the capture agent in the capture region.

As described above, the fluid sample 111 solubilizes the first complex 121, the second labeled antibody 123, and the third labeled antibody 122. In one implementation, during the process, first target analyte 112 in sample 111 does not interact, or does not substantially interact, with first complex 121. Without being bound by any particular theory, in this implementation of the lateral flow devices described herein, the first analyte of interest 112 is not conjugated, bound, or associated with the first complex 121 as the sample 111 flows through the label zone 120. In another implementation of the lateral flow device described herein, when the fluid sample 111 dissolves the first complex 121, the first target analyte 112 in the sample 111 interacts with the first complex 121. In one non-limiting embodiment, and without being bound by any particular theory, at least some of the first analyte of interest 112 in the sample 111 is exchanged with the first analyte present in the first complex 121. Without being bound by any particular theory, in this implementation, the first capture agent 136 in the first capture zone 135 can bind to at least some of the first complexes 121, wherein the analyte in the first complexes 121 is the first target analyte 112 introduced onto the device 101 via the sample 111.

As shown in fig. 2A and 2B, when first, second, and third target analytes 112, 113, and 114, respectively, are not present in the fluid sample 111 (or are present at less than detectable levels), the first complexes 121 saturate the first capture agents 136 at the first capture zones 135 (e.g., each molecule of the first capture agent 136 in the first capture zones 135 binds to one first complex 121 flowing out of the label zone 120). The second capture agent 134 in the second capture area 133 does not bind to any second complex because a second complex does not form in the absence of the second target analyte 113. In the event that the second target analyte 113 is present at less than detectable levels, no detectable amount of the second complex is formed. The third capture agent 132 in the third capture area 131 does not bind to any third complexes because third complexes do not form in the absence of the third target analyte 114. In the event that the third target analyte 114 is present at less than detectable levels, no detectable amount of the third complex is formed. The first complex 121 captured in the first capture area 135 emits a first detectable light signal that is the maximum intensity signal that can be obtained from the first capture area 135 of the lateral flow device 101. In scenarios where the first target analyte 112 is not present (or is present at less than a detectable level) in the sample 111, the first optical signal detected at the first capture zone 135 is the maximum intensity signal at the first capture zone, since each available first capture agent 136 has bound to the first complex 121 at the first capture zone 135. In the absence (or less than the detectable level) of the second target analyte 113, no second complex is formed (or no detectable amount of the second complex is formed), and thus no second complex (or any detectable amount of the second complex) is captured by the second capture agent 134 and no second detectable signal is observed. Similarly, in the absence (or less than a detectable level) of the third target analyte 114, no third complex is formed (or no detectable amount of the third complex is formed), and thus no third complex (or any detectable amount of the third complex) is captured by the third capture agent 132 and no third detectable signal is observed.

Fig. 3A-3B illustrate exemplary lateral flow assays where only the first target analyte 112 is present in the fluid sample 111 and the second 113 and third 114 target analytes are not present in the fluid sample 111 or are present below detectable levels. In this example, the first target analyte 112 competes with the first complex 121 for binding to the first capture agent 136 at the first capture zone 135. The result is that as the concentration of first target analyte 112 in sample 111 increases, the amount of first target analyte 112 bound by first capture agent 136 at first capture zone 135 increases. Because the first target analyte 112 does not emit a detectable signal, and because less of the first complex 121 binds to the first capture agent 136 at the first capture zone 135 in the presence of the first target analyte 113, the first detectable signal is reduced compared to the maximum signal intensity observed when the first target analyte 112 is not present in the sample 111.

An exemplary dose response curve depicting the example lateral flow assay of fig. 3A and 3B is shown in fig. 7A. In fig. 7A, the signal intensity of the first target analyte detected at the first capture zone (where the signal intensity measured by the first capture zone configured to bind CRP is plotted as a square) decreases with increasing concentration of the first target analyte in the sample. In contrast, because the second target analyte and the third target analyte are not present (or less than a detectable level) in the sample, the second signal of the second target analyte (where the signal intensity measured by the second capture zone configured to bind IP-10 is plotted as a triangle) and the third signal of the third target analyte (where the signal intensity measured by the third capture zone configured to bind TRAIL is plotted as a circle) do not increase.

Fig. 4A-4B illustrate example lateral flow assays where only the second target analyte 113 is present in the fluid sample 111 and the first and third sample target analytes 112 and 114 are not present in the fluid sample 111 or are present below detectable levels. In this example, the second target analyte 113 binds to a second labeled antibody 123 that specifically binds to the second target analyte 113, forming a second complex. The second complex flows with the fluid sample 111 to the detection zone 130 where it is bound by the second capture reagent 134 at the second capture zone 133. A second detectable signal is emitted from the bound second complex at the second capture area 133, indicating the presence of the second target analyte 113 in the fluid sample 111. As the concentration of the second target analyte 113 in the sample 111 increases, the intensity of the second detectable signal emitted by the second complex bound by the second capture area 133 increases.

An exemplary dose response curve depicting the example lateral flow assay of fig. 4A and 4B is shown in fig. 7B. In fig. 7B, the signal intensity of the second target analyte (here, the signal intensity measured by the second capture zone configured to bind IP-10 is plotted as a triangle) increases with increasing concentration of the second target analyte in the sample. The signal intensity of the first target analyte (here, the signal intensity measured by the first capture zone configured to bind the CRP is plotted as a square) remains or substantially remains at a maximum value (in this example, about 70AU (arbitrary signal intensity units)) for all concentrations of the second target analyte, indicating that the first target analyte is not present in the sample (or is less than a monitorable level). The signal intensity of the third target analyte (here, the signal intensity measured by the third capture zone configured to bind TRAIL is plotted as a circle) does not increase, indicating that the third target analyte is not present in the sample (or is less than a monitorable level).

Fig. 5A-5B illustrate an example lateral flow assay in which only third target analyte 114 is present in fluid sample 111, while second sample target analyte 113 and first target analyte 112 are absent or present below detectable levels in fluid sample 111. In this example, the third analyte of interest 114 binds to a third labeled antibody 122 that specifically binds to the third analyte of interest 114, forming a third complex. The third complex flows with the fluid sample 111 to the detection zone 130 where the third complex is bound by the third capture agent 132 at the third capture zone 131. A third detectable signal is emitted from the bound third complex at the third capture area 131, indicating the presence of the third target analyte 114 in the fluid sample 111. As the concentration of the third target analyte 114 in the sample 111 increases, the intensity of the third detectable signal emitted by the third complex bound by the third capture area 131 increases.

An exemplary dose response curve depicting the example lateral flow assay of fig. 5A and 5B is shown in fig. 7C. In fig. 7C, the signal intensity of the third target analyte (here, the signal intensity measured by the third capture zone configured to bind TRAIL is plotted as a circle) increases with increasing concentration of the third target analyte in the sample. The signal intensity of the first target analyte (here, the signal intensity measured by the first capture zone configured to bind the CRP is plotted as a square) remains or substantially remains at a maximum value (in this example, about 70AU) for all concentrations of the third target analyte, indicating that the first target analyte is not present (or less than a monitorable level) in the sample. The signal intensity of the second target analyte (here, the signal intensity measured by the second capture zone configured to bind IP-10 plotted as a triangle) does not increase, indicating that the second target analyte is not present in the sample (or is less than a monitorable level).

Fig. 6A-6B illustrate an example lateral flow assay in which only first 112 and second 113 target analytes are present in the fluid sample 111 and third target analyte 114 is not present in the fluid sample 111 or is present below a detectable level. The example lateral flow assay is a combination of fig. 3A and 3B and fig. 4A and 4B, illustrating an iteration in which more than one target analyte may be present but not necessarily all target analytes are present (or not necessarily present at detectable levels). In this example, the first target analyte 112 in the sample competes with the first complex 121 for binding to the first capture agent 136 at the first capture zone 135 in the manner described above with reference to fig. 3A and 3B. The first detectable signal detected at first capture zone 135 decreases from a maximum signal intensity as the concentration of first target analyte 112 increases, indicating the presence and quantity of first target analyte 112 in fluid sample 111. At or near the same time, a second target analyte 113 binds to a second labeled antibody 123 in the label zone to form a second complex. The second complex flows to the detection zone and binds to the second capture agent 134 at the second capture zone 133. The second detectable signal increases as the concentration of the second target analyte 113 increases, indicating the presence or quantity of the second target analyte 113 in the fluid sample 111.

Fig. 1A-6B illustrate a first capture area 135, a second capture area 133, and a third capture area 131 arranged perpendicular to the longitudinal axis of the test strip, where the first capture area 135 is furthest from the sample receiving area 110 and the third capture area is closest to the sample receiving area 110. In this non-limiting example, the first complex 121 will flow through the third capture area 131 and the second capture area 133 before reaching the first capture area 135 and binding the first capture agent 136 immobilized at the first capture area 135. These figures are illustrative and various iterations, changes and modifications may be implemented. The relative positions of the first 135, second 133 and third 131 capture zones may be different from those illustrated in fig. 1A-6B, such that the fluid sample 111 flows through the capture zones in a different order than that shown. For example, the first capture area, the second capture area, and the third capture area can be arranged in various orders (e.g., 3, 2, 1; 3, 1, 2; 1, 2, 3; 1, 3, 2; 2, 1, 3; or 2, 3, 1) perpendicular to the longitudinal axis of the test strip. Furthermore, the capture zones may be positioned parallel to, rather than perpendicular to, the longitudinal axis of the test strip such that each capture zone is equally spaced from the sample receiving zone.

There are many ways to determine the maximum intensity signal of the first capture area 135 of the lateral flow device 101. In one non-limiting embodiment, the maximum intensity signal obtained from a particular first capture area 135 of the lateral flow device 101 may be determined empirically and stored in a look-up table. In some cases, the maximum intensity signal is determined empirically by testing a lateral flow device 101 having known characteristics and configurations, for example, by averaging the maximum intensity signals obtained when applying a sample of the first target analyte at zero or nearly zero concentration to a lateral flow device 101 of known dimensions and configuration. In another non-limiting embodiment, theoretical calculations of known specifications and configurations (such as, for example, the number and specific characteristics of the first complex 121 integrated on the marker region 120) for a given lateral flow device 101 can be used to determine the maximum intensity signal that can be obtained from a particular first capture region 135 of the lateral flow device 101.

Further, it should be understood that although reference is made herein to a "maximum intensity signal," a signal within a particular range of expected maximum intensities may be considered to be substantially equal to a "maximum intensity signal. Additionally, it should be understood that "maximum intensity signal" may refer to a maximum intensity optical signal, a maximum intensity fluorescent signal, a maximum intensity magnetic signal, or any other type of signal that occurs at a maximum intensity. As one non-limiting example, a detected signal at first capture area 135 that is within 1% of the expected maximum intensity signal is considered to be substantially equal to the expected maximum intensity signal at first capture area 135. If the maximum intensity signal is at or about 70AU, a detected signal in the range of about 75.3AU to about 70.7AU will be considered to be substantially equal to the maximum intensity signal of 70 AU. As another example, in the non-limiting embodiment described with respect to fig. 7A-7C, the detected signal at first capture area 135 within 10% of the expected maximum intensity signal is considered to be substantially equal to the expected maximum intensity signal at first capture area 135. Thus, in the example illustrated in fig. 7A-7C where the maximum intensity signal is at or about 70AU, a detected signal in the range of about 63AU to about 77AU is considered to be substantially equal to the maximum intensity signal of 70 AU. These examples are provided for illustrative purposes only, so other deviations may be acceptable. For example, in a lateral flow assay device according to the present disclosure, a detected signal at first capture zone 135 within any suitable range of deviation from an expected maximum intensity signal (such as, but not limited to, within 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 2.0%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15% of the expected maximum intensity signal) can be considered to be substantially equal to the expected maximum intensity signal at first capture zone 135.

As illustrated in fig. 7A, in embodiments of lateral flow devices according to the present disclosure, it is advantageous for the first signal at first capture area 135 to gradually decrease as the concentration of the first target analyte increases. As a result of this gradual decrease in the detected first signal, embodiments of the lateral flow device described herein advantageously allow the detector to accurately measure the first signal at high resolution and the data analyzer to determine the concentration of the first target analyte at high concentrations with high accuracy.

In addition, according to the present disclosure, a dose response curve for a target analyte present at high concentration in a lateral flow device advantageously begins with a maximum intensity signal and then decreases from the maximum intensity signal. This means that advantageously, in the dose response curve of the first analyte present in high concentration, there will be no signal of the same amplitude as the maximum intensity signal in the portion of the dose response curve where the signal is reduced. Further, because the first signal will be the same or effectively the same as the maximum intensity signal at low concentrations of the first analyte in the sample (e.g., they are considered to be substantially equal to the maximum intensity signal, as described above), there is a plateau of the first optical signal at a relatively constant value ("maximum intensity signal") for zero to low concentrations of the first analyte (as will be discussed in detail below with reference to non-limiting examples). This means that advantageously, in the part of the first dose-response curve where the first signal decreases, there will be no signal with about the same amplitude as the maximum intensity signal. Thus, with respect to analytes present at high concentrations in embodiments of the lateral flow devices described herein, false negatives and inaccurate low readings are avoided, and allow for the detection of both high and low concentrations of analytes present in a single sample without the need for dilution or other pre-treatment of the sample prior to application to the single lateral flow assay.

Advantageously, in the embodiments of the lateral flow devices described herein, the first complex 121 can be pre-formulated to include a known amount of the first analyte of interest and then deposited on the conjugate pad. In some embodiments, a known concentration of a first analyte of interest is incubated with an antibody or antibody fragment and a marker molecule in a reaction vessel separate from the test strip. During incubation, the first analyte of interest is conjugated to, bound to, or otherwise associated with the antibody and label molecule, thereby forming the first complex 121 described above. After incubation, the first complex 121 is added directly to the solution at a precisely known concentration or separated to remove excess free first target analyte, and then sprayed onto the conjugate pad. A solution including the first complex 121 is applied to a test strip, such as the label region 120 described above. During deposition, the first complex 121 is integrated on the surface of the test strip. In one non-limiting embodiment, the first complex 121 is integrated onto the conjugate pad of the test strip. Advantageously, the first complex 121 may remain physically bound to and chemically stabilized on the surface of the test strip until the operator applies the fluid sample to the test strip, whereupon the first complex 121 separates from the test strip and flows with the fluid sample, as described above.

Similarly, the second labeled antibody 123 and the third labeled antibody 122 can be formulated separately. For example, a second antibody that specifically binds a second target analyte can be incubated with a marker molecule, thereby forming a second labeled antibody 123. The second labeled antibody 123 may be deposited on the test strip similar to the deposition of the first complex 121, or in any other suitable manner. The second labeled antibody 123 may remain physically bound to and chemically stabilized on the surface of the test strip until the operator applies the fluid sample to the test strip whereupon the second labeled antibody 123 separates from the test strip, binds with any second analyte present in the fluid sample and flows with the fluid sample, as described above. Similar methods may be used for the third labeled antibody, or any additional labeled antibody or complex, for detecting additional target analytes.

In some embodiments, first complex 121, second labeled antibody 123, and third labeled antibody 122 are each deposited in an amount ranging from about 0.1-20 μ L per test strip. In some embodiments, the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 are each deposited in the label zone in the following amounts: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μ L/test strip. In one non-limiting example, the first complex 121 is deposited in an amount of about 3 μ L/cm, the second labeled antibody 123 is deposited in an amount of about 7 μ L/cm, and the third labeled antibody 122 is deposited in an amount of about 7 μ L/cm.

The solution comprising the first complex 121, the solution comprising the second labeled antibody 123, and the solution comprising the third labeled antibody 122 can be applied to the test strip in a number of different ways. In one example, the solution is applied to the marker region 120 by spraying the solution using an air-jet technique. In another example, the solution is deposited by pouring the solution, spraying the solution, formulating the solution into a powder or gel solution that is placed or rubbed on the test strip, or any other suitable method to apply the first complex 121, the second labeled antibody 123, and the third labeled antibody 122. In some embodiments, after deposition, the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 are dried on the surface of the test strip after deposition by heating or blowing on the conjugate pad. Other mechanisms of drying the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 on the surface of the test strip are suitable. For example, vacuum or lyophilization may also be used to dry the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 on the conjugate pad.

In some cases, the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 are not added to the solution prior to deposition, but are applied directly to the test strip. The first complex 121, the second labeled antibody 123, and the third labeled antibody 122 may be applied directly using any suitable method, including but not limited to applying compressive or vacuum pressure to the first complex 121, the second labeled antibody 123, and the second complex 121 on the surface of the test strip and/or applying the first complex 121, the second labeled antibody 123, and the third labeled antibody 122 in lyophilized particulate form to the test strip surface.

Embodiments of the lateral flow assay described herein need not include a control line or zone configured to confirm that the applied sample in the sample receiving zone 110 has flowed to the detection zone 130 as expected. Under normal operating conditions, some detectable signal will always be emitted from the first capture area 135 if the sample has flowed to the first capture area 135. Advantageously, the first capture zone 135 may be positioned downstream of both the third capture zone 131 and the second capture zone 133 and may visually indicate that the sample 111 has flowed through all three capture zones as intended, such that the first capture zone 135 effectively functions as a control line or zone. Under normal operating conditions, if the sample has flowed to the first capture area 135, a detectable signal will always be emitted from the first capture area, even if the first target analyte is present in the sample at a very low concentration. This is because the lateral flow device of the present disclosure produces a first dose-response curve that is still at or near the maximum intensity signal for zero or low concentrations of the first target analyte. By careful design of the lateral flow assay, the signal at first capture area 135 can be significantly reduced, but not completely eliminated, even in the presence of a physiologically possible high concentration of first analyte 112 in the sample. Thus, the absence of any detectable signal at the first capture zone 135 after a sample has been applied to the sample receiving zone 110 can be used to indicate that the lateral flow assay is not operating as intended (e.g., the sample does not flow as intended to the first capture zone 135, or as another example, the first capture agent 136 immobilized at the first capture zone 135 is defective or faulty). Thus, a further advantage of embodiments of lateral flow devices according to the present disclosure is the ability of the first capture zone 135 to function as a control line thereby allowing for the complete omission of a separate control line from the test strip. However, it should be understood that a control line may be included in embodiments of the lateral flow devices described herein for a variety of purposes, including but not limited to viewing a line, for normalizing noise, or for detecting interference of an analyte in serum.

In some cases, the lateral flow device includes one or more control zones. The control zone may be at or separate from the detection zone. In some embodiments, the control zone can be a positive control, which can include a small molecule conjugated to a protein, such as Bovine Serum Albumin (BSA). An antibody that specifically binds to a positive control marker for the small molecule can be deposited on the conjugate pad. When the positive control labeled antibody is rehydrated with the liquid sample, it flows toward the positive control zone and binds to the small molecule, forming a half-sandwich. The positive control signal generated at the positive control zone is independent of the presence or concentration of the plurality of target analytes present in the fluid sample and thus maintains a relatively constant intensity. However, due to the difference in the amount of positive control labeled antibody deposited on the conjugate pad caused by the non-uniform pad material, the intensity of the positive control signal generated at the positive control zone and the intensity of the signal generated at each capture zone can differ slightly from device to device, even if the same sample is tested. The intensity variation of the signal is the same between the devices for the positive control zone and the capture zone. Thus, the positive control zone can be used as a reference line to better measure the relative signal intensity generated at the capture zone and thus the positive control zone can provide a more accurate analyte concentration.

The lateral flow assay may additionally include a negative control zone. The negative control zone may include a negative control antibody from the same species as the antibody used in the capture zone. Some components from some blood samples may interfere with immunoassays. If such interfering substances are present in a sample, they will interfere not only with the signal intensity at the capture zone, but also with the signal intensity at the negative control zone. Embodiments of the reader and data analyzer disclosed herein can process signal measurements obtained from the negative control zones to correct any calculations or alert the operator of invalid results.

The following non-limiting examples illustrate features of the lateral flow devices, test systems, and methods described herein, but are in no way intended to limit the scope of the present disclosure.